O-linked beta-N-acetylglucosamine (O-GlcNAc) is a dynamic and inducible post-translational modification that is covalently attached to serine and threonine residues on nuclear and cytoplasmic proteins. We and others have demonstrated that elevation in the levels of O-GlcNAc transferred to intracellular proteins induces insulin resistance, the hallmark of Type II diabetes. The specific aims of this proposal will test the hypothesis that insulin action is inhibited by elevated O-GlcNAc modification of specific proteins in the insulin signal transduction cascade. In 3 different model systems, we have determined that the defect lies in the metabolic branch of insulin signaling, downstream of the receptor and at or upstream of the protein kinase AKT. We have developed several essential tools for the study of the O-GlcNAc posttranslational modification in insulin signal transduction. These include an O-GlcNAc specific monoclonal antibody, cell lines for the inducible expression of enzymes that add and remove O-GlcNAc, C. elegans strains that harbor deletions in the O-GlcNAc cycling enzymes, and a tandem mass spectrometry-based approach for site-mapping and quantification of post-translational modifications. In Specific Aim 1, we will expand on our preliminary findings to demonstrate that inappropriate O-GlcNAc modification perturbs insulin signaling as measured by changes in apoptosis, glucose-uptake, and/or lifespan using mammalian cell lines and C. elegans as model systems. Furthermore, functional changes (activity, localization, associations, and phosphorylation) in proteins of the metabolic branch of the insulin pathway upon perturbations in global O-GlcNAc levels will be elucidated. In Specific Aim 2, we will identify and sitemap O-GlcNAc modified proteins associated with the insulin signaling pathway using our suite of tandem mass spectrometry-based approaches. In Specific Aim 3, we will assign functional consequences to O-GlcNAc modification of specific signal transduction proteins in the metabolic branch of the insulin cascade. By reintroducing tagged glycosylation-competent wild type and glycosylation-incompetent mutant proteins back into mammalian cell lines and into C. elegans strains that are null for the protein of interest, we will determine how differential O-GlcNAc modification of specific sites on particular proteins affects insulin signaling. Completion of these aims will elucidate both global and molecular consequences of protein O-GlcNAc modification with regard to regulating insulin action in mammalian cell lines and in C. elegans. [unreadable] [unreadable] [unreadable]